Avidin is a basic glycoprotein derived from egg white and strongly binds to biotin (vitamin H). Streptavidin is an avidin-like protein derived from Actinomycetes (Streptomyces avidinii) and has a near-neutral isoelectric point and does not have a sugar chain. Both proteins form tetramers, and the molecular weights thereof are about 60 kDa. The tetramer is formed by weak bonds between dimers, while the dimers are composed of strongly bonded monomers. Avidin and streptavidin have the property that one monomer thereof binds to one biotin molecule. Avidin and streptavidin each have significantly high affinity (Kd=10−15 to 10−14 M) to biotin, and the affinity is one of the most strong interactions between two biomolecules. Accordingly, avidin/streptavidin-biotin interaction has been widely used in the fields of biochemistry, molecular biology, and medicine.
Biotin has a small molecular weight of 244 and is stable to a change in pH and heat and, therefore, is commonly used for labeling substances. In a method of biotinylation, chemically modified biotin is bound to a functional group of a target compound. Such biotinylating reagents are commercially available and can be used to biotinylate compounds such as protein and nucleic acid. In one of the methods of biotinylation of proteins, a fusion protein of a target protein and a sequence that can be biotinylated by biotin ligase in a living body is expressed as a recombinant protein, and the resulting fusion protein can be biotinylated by the biotin ligase in a host cell.
The present inventors have discovered tamavidin 1 and tamavidin 2, which are novel avidin-like biotin-binding proteins, in an edible mushroom, Pueurotus cornucopiae (WO02/072817). Tamavidin 1 and tamavidin 2 can be expressed in a large amount in Escherichia coli. In particular, tamavidin 2 can be easily prepared by purification using an iminobiotin column (WO02/072817). Tamavidin 1 and tamavidin 2 form tetramers and form an extremely strong binding with biotin. Furthermore, tamavidin 2 is an excellent biotin-binding protein in that the protein exhibit a heat resistance higher than that of avidin or streptavidin and that the non-specific binding is less than that of avidin.
Avidin, streptavidin, and tamavidin have higher heat resistance than normal protein and have heat resistance determined by a method using fluorescent biotin (expressed by a temperatures at which their activity decreases to one half the initial activity) of 79° C., 74° C., and 85° C., respectively (Takakura et al. 2009 FEBS J 276: 1383-1397).
For expanding industrial applications, however, attempts to further enhance the heat resistance of avidin and streptavidin have been being made. Reznik et al., (1996) reported streptavidin (Nat. Biotechnol., 14: 1007-1011). Aiming to strengthen the weak bond between the dimers of streptavidin, they mutate a hystidine (His) residue at position 127 into cysteine (Cys) through genetic engineering to construct a heat-resistant disulfide-linked dimer of streptavidin mutants. The mutant maintained about 70% of the original biotin-binding activity after a heat treatment at 90° C. for 10 min, while wild-type streptavidin maintained about 55% of the original biotin-binding activity after a heat treatment at 70° C. for 10 min.
Meanwhile, Nordlund et al. 2003 (J. Biol. Chem. 278: 2479-2483) reported heat stabilization of avidin; they have genetically engineered various forms of disulfide bonds between avidin subunits to increase heat resistance. The residual biotin-binding activity of avidin was almost zero after a treatment at 99.9° C. for 2 min, while the activity of I117C (modified avidin in which isoleucine at position 117 in avidin is replaced with cysteine) was a little more than 30%, and the activity of D86CI106CI117C (modified avidin in which aspartic acid residue at position 86 and isoleucine residues at positions 106 and 117 in avidin are replaced with cysteine residues, respectively) was a little less than 50%.
Furthermore, Hytonen et al., (2005), J. Biol. Chem., 280: 10228-10233 reported heat stabilization of avidin without the formation of disulfide bonding. A chimera of avidin and AVR4 (protein encoded by Avidin-related gene 4) having higher heat resistance than avidin was formed to create an avidin mutant ChiAVD (I117Y), which exhibited increased residual biotin-binding activity of up to 98% after a treatment at 99.9° C. for 32 min (residual activity: 4% for avidin, 72% for AVR4).
Almost all of the heat-resistant avidin mutants are prepared using an insect cell expression system using baculovirusm, while streptavidin mutants are prepared using an E. coli expression system, which requires a step of solubilization from insoluble inclusion bodies during the process. Thus, such proteins as described above, any of which require considerable costs and efforts for manufacturing them, have not yet been put into practical application.